University of Southern California Dissertations and Theses

Investigating the role of cardiac fibroblasts in modulating angiogenesis during heart injury

(USC Thesis Other)

Investigating the role of cardiac fibroblasts in modulating angiogenesis during heart injury

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

Investigating the Role of Cardiac Fibroblasts in Modulating
Angiogenesis During Heart Injury

By

Aesha Upadhyay

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)

August 2020

Copyright 2020 Aesha Upadhyay
ii

Acknowledgements

I would like to express my sincere gratitude to my advisor, Dr. Jian Xu for giving me the
opportunity to work on this project and for her constant guidance and encouragement. I
am also very grateful to Dr. Young Kwon-Hong and Dr. Ching-Ling Lien for being on my
thesis committee and providing me with immensely helpful suggestions and feedback.
I would like to thank my mentor, Dr. Jiang Qian, for teaching me all the techniques and
helping me navigate through my project. I am also very thankful to CCMB and all my lab
members for their constant help and support throughout my time in the Xu Lab.
I would also like to extend my special gratitude to my friends and family for being my
strongest support system and motivating factor.

iii

Table of Contents

Acknowledgements……………………………………………………………………………...ii
List of Figures…………………………………………………………………………...……....iv
Abstract ……………………………….…………………………………………...…...………..v
Chapter 1: Introduction………………………..………………………….………………....….1
1.1 Heart Disease…………………………...……………………………………….….1
1.2 Left ventricular hypertrophy………………………………………………………..2
1.3 Cardiac fibrosis…………………….…………………………………....................4
1.4 Contribution of coronary vasculature………………………………………...…...7
1.5 Angiogenic Insufficiency…………….……………………………………...……...8
1.6 Disease model………………………………………………………….…………...9
Hypothesis…………………………………………………………………..………………….11
Objective of the Study………………………...…………………………….………………...11
Chapter 2: Materials and Methods……………………………………...…………………...12
2.1 DNA Genotyping………………………………………………..……………….12
2.2 Surgery …………………………………………………………………………..13
2.3 Tissue harvest and processing………………………………………………...15
2.4 Histological Analysis…………………………………………………………….16
2.5 Cell culture……………………………………………………………………….17
2.6 Fibrin gel bead assay………………………………...…………………………17
Chapter 3: Results……………………………………………………………………………..22
Discussion………………………………………………………………..………………...…. 26
References……………………………………………………………………………………. 29

iv

List of Figures

1.2 Ventricular hypertrophy in a failing heart………………………………………………...2

1.3a Activation of fibroblasts post or during an injury……………………………….………4

1.3b Crosstalk between cardiac tissue cell types…………………………….…..…….…...6

1.5 Increased inter-capillary distance in a hypertrophic heart…..………………..…..……8

1.6a Transgenic models …………………………………………………..…..……………….9

1.6b Mechanism of action of isoproterenol drug …………………………………………..10

2.8a Schematic diagram of fibrin gel bead assay……………………………………….....17

2.8b HUVEC coated cytodex-3 microcarrier bead ………………………………………...20

2.8c Fibrin gel embedded with HUVEC coated microcarrier beads ……………………..21

3.1 YFP – αSMA co-staining ……………………………………………………...………....22

3.2 Picrosirius red staining …………………………………………………………………...23

3.3 CD31 marker staining …………….………………………………………………….......24

3.4 Heart weight / Body weight bar graph ………………………………………...………..25
v

Abstract

Diseases like hypertension, myocardial ischemia, diabetes, congenital heart defect
etc. worsen with time and make the heart work harder for it to supply enough blood
throughout the body. This excess workload causes injury to the cardiac tissue and the
heart muscle undergoes hypertrophy to compensate for its loss of function. The
pathological cardiomyocyte growth demands increase in nutrient and oxygen supply
which is not always backed up by the proportional increase in angiogenesis. This lack of
vascularization worsens cardiac remodeling and tissue contractility during injury and
accelerates the progression to heart failure.
Recent studies have found the paracrine roles of cardiomyocytes, pericytes and
inflammatory cells in (dys)regulating angiogenesis during cardiac hypertrophy, but little is
known about the role of cardiac fibroblasts in this process. Cardiac fibroblasts are
responsible for providing support and maintaining the structure of the heart tissue by
secreting extracellular matrix (ECM) proteins. These resident fibroblasts become
activated and transform into myofibroblasts to secrete excess ECM proteins during the
wound repair process post injury (Travers et al., 2016).
My project focuses on understanding the role of resident and activated fibroblasts
in modulating angiogenesis during cardiac injury. I will be using in vivo and in vitro models
to investigate this question.

1

Chapter 1: Introduction
1.1 Heart Disease
Cardiovascular diseases (CVDs) are the leading cause of death globally. This group
of diseases include disorders of the heart and blood vessels such as coronary heart
disease, cerebrovascular disease, rheumatic heart disease etc. (Mensah & Brown, 2007)
Among these, the heart diseases alone are responsible for 1 in every 4 deaths in the
United States, making them the nation’s number one cause of mortality. These diseases
not only impact the quality of life at large but also impose great economic burden. In the
United States, about $219 billion are spent each year on health care services, medicines
and lost productivity due to heart disease related deaths.
Several heart conditions worsen with time and advance to heart attack or heart failure.
Heart attacks are a result of blocked arteries that stop the blood flow to the heart, leading
to tissue death from lack of oxygen supply. On the other hand, heart failure occurs when
the heart is not able to fill enough blood (diastolic heart failure) or pump the blood with
enough force (systolic heart failure) for its supply to the rest of the body (Roger, 2013). It
is a progressive condition that occurs due to diseases that damage the heart, like
myocardial ischemia, high blood pressure, diabetes etc. Heart failure exclusively
contributes to 1 in 8 deaths in the United States, costing about $30.7 billion in a year.
Currently, no cure has been found, but advancements in treatment options can help
patients live longer and more active lives.

2

1.2 Left Ventricular Hypertrophy

Figure 1.2 Ventricular hypertrophy in a failing heart (Adapted from Cardiosecur-heart
failure)
Diseases like high blood pressure cause narrowing of the arteries and require the
heart muscle to work harder to pump blood efficiently throughout the body. Similarly,
myocardial ischemia and diabetes decrease the blood supply to the heart muscle, forcing
it to work more in order to maintain its function. To adapt to this workload, the heart wall
thickens, especially of the left ventricle which is responsible for pumping blood to the
entire body. The thickening refers to increase in area of individual cardiac myocytes as
new sarcomeres are added within the cells. This enlargement of the chamber walls in
3

response to increased volume load or pressure overload is termed as left ventricular
hypertrophy. Hypertrophy (or concentric hypertrophy) is one of the two distinct
morphological alterations that can occur in a failing heart, where the other being
ventricular dilation (eccentric hypertrophy) that is induced by volume overload
(Grossman, 1980). Both these adaptations to reduced heart function are referred to as
cardiac remodeling. Here, we are only focusing on the hypertrophic ventricular
remodeling. Other diseases such as congenital heart defect, heart valve disease etc. also
develop left ventricular hypertrophy to overcome heart’s functional inefficiency.
Heart failure comprises of compensation and/or decompensation phase(s). A
hypertrophied heart is able to make up for its weakened pumping action in the
compensation phase. This stage eventually progresses to decompensation when the
chamber walls undergo cardiac dilatation or become too thick to be able to contract and
relax efficiently. This further deteriorates the heart’s condition and aggravates quickly to
end stage heart failure where the heart muscle completely ceases to function (Opie,
1990).

4

1.3 Cardiac Fibrosis

Figure 1.3a Activation of fibroblasts into myofibroblasts in a hypertrophic heart post or
during an injury (Herum et al., 2017)

Cardiac Fibrosis is another major factor of pathological cardiac remodeling that
concerns almost all forms of heart diseases. In response to an injury, the resident
fibroblasts within the heart become activated and transform into the myofibroblast
phenotype. These myofibroblasts then secrete and deposit excessive extracellular matrix
proteins to maintain the structural integrity and function of the injured heart muscle
(Travers et al., 2016).
The localization of collagen and other matrix proteins largely depends on the type of
injury that has occurred. For diseases such as myocardial infarction and ischemia, injury
is primarily in the form of cardiomyocyte death. In such cases, collagen deposition forms
a scar at the site of injury and prevents the heart tissue from rupturing. This type of
reparative response to cardiomyocyte death is termed as replacement fibrosis. Usually,
5

after secretion of collagen, the myofibroblasts undergo apoptosis and die. Though, if the
process continues, the pro-inflammatory and pro-hypertrophic signals along with collagen
secretion also affects the healthy tissue that is far from the site of injury. This kind of
interstitial and/or perivascular fibrosis which might not be directly related to cardiomyocyte
death is defined as reactive fibrosis. Reactive fibrosis is also seen in response to
pathological stimuli that affects heart’s pumping action such as pressure overload, volume
overload, metabolic dysfunction etc. (González et al., 2018).
The initial purpose of fibrosis is to help in wound healing, but with time it causes the
heart muscle to become stiff and further worsens the tissue morphology and pumping
action. Thus, pathological deposition of collagen and other matrix proteins poses a major
threat to the viability of the organ during acute injuries.

6

Figure 1.3b Crosstalk between cardiac tissue cell types in activating fibroblasts and
regulating angiogenesis (Alex & Frangogiannis, 2018)

Cardiac fibroblasts responsible for this cardiac remodeling, make up to 20% of the
cardiac cell population. It is also widely known that fibroblasts are needed for
angiogenesis, especially, for lumen formation. Upon injury, the hypertrophied
cardiomyocytes along with pericytes and inflammatory cells, secrete profibrotic factors
such as TGFβ that stimulates the fibroblasts to become activated into myofibroblasts.
These differentiated fibroblasts within the tissue possess enhanced migratory, contractile
and ECM producing capabilities. Myofibroblasts are present only in an injured tissue and
are responsible for wound repair process. Prolonged injury prevents myofibroblasts from
undergoing apoptosis or reverting to its inactivated state. They secrete excess
extracellular matrix components such as fibronectin and collagen that serve as a barrier
7

in electrophysiological stimuli conduction between cardiomyocytes and hamper their
beating action. As previously mentioned, a lot is known about the role of other cardiac
cell types in promoting angiogenesis during pathological hypertrophy, but not enough is
known about the role of fibroblasts and myofibroblasts in this process (Travers et al.,
2016) (Davis & Molkentin, 2014).

1.4 Contribution and Regulation of Coronary Vasculature During Heart Failure
The normal functioning of the heart muscle is closely connected to the coronary blood
flow and oxygen supply. The left ventricle, responsible for pumping blood to the entire
body, utilizes most of this oxygen to sustain its function. During pathological hypertrophy,
the distance between capillaries surrounding the cardiomyocytes increases, making it
difficult for the cells to access enough oxygen. The coronary vessels dilate to increase
the blood supply and meet with the increased oxygen demand of the hypertrophied
myocytes. This vasodilation is regulated by several metabolic, neurohumoral and
mechanical influences as well as paracrine factors secreted by erythrocytes, platelets and
smooth muscle cells. The compensatory dilation of vessels is beneficial for the
cardiomyocytes up to a certain extent beyond which the need for new vessels increases
within the tissue. Promoting angiogenesis in a hypertrophic heart is shown to improve the
its function and delay heart failure (Duncker et al., 2015).

8

1.5 Angiogenic Insufficiency During Pathological Hypertrophy

Figure 1.5 Angiogenic insufficiency and increased inter-capillary distance in a
hypertrophic heart (de Graaf et al., 2014)

Angiogenesis is referred to the formation of new blood vessels from the existing ones.
Coronary Angiogenesis is a process crucial in maintaining vessel density and oxygen
supply during physiological growth and pathological remodeling of the heart. Though,
when the heart muscle undergoes pathological hypertrophy, its increased need of energy
supply is not always backed up by the proportional increase in angiogenesis. This lack of
vascularization exacerbates pathological remodeling and contractile dysfunction and
accelerates the progression to heart failure.
Several anti-angiogenic factors such as TGFβ, angiostatin, thrombospondin-1/-2 etc.
are found to be upregulated during pathological hypertrophy. Recent studies have
Hypertrophy
9

investigated the role of cardiomyocytes, endothelial cells, pericytes as well as
inflammatory cells, and the factors expressed by them in promoting angiogenesis during
cardiac hypertrophy. Clinical significance of these factors has been tested and they’ve
not yet been established as potential treatment options. Fibroblasts are known to express
a few factors similar to cardiomyocytes and endothelial cells such as VEGF and PDGF
and are known to support lumen formation. They are also known to secrete
thrombospondin, an angiogenic inhibitor during the hypertrophy stage. This knowledge
on fibroblasts is not enough to efficiently tackle this major pathological issue of angiogenic
insufficiency and hence, more investigation is needed (Gogiraju et al., 2019).

1.6 Disease Model:

i) Control ii) Experimental

Figure 1.6a Transgenic models i) Cre driven by Periostin promoter for YFP reporter
expression. ii) Cre driven by Periostin promoter for DTA and YFP expression

C57BL/6 mice from Jackson Laboratories were used to generate an animal model for
this project. Homozygous Postn-MerCreMer (Postn-MCM) and DTA mice were bred with
10

homozygous YFP mice to obtain heterozygous Postn-cre/+;DTA/+;YFP/+
(Pn
MCM
;ROSA
YFP
;ROSA
DTA
) colony for the experimental group. Here, the DTA gene
codes for Diphtheria Toxin fragment A which is regulated under the Periostin (Postn/Pn)
promoter (Kaur et al., 2016). Postn is an exclusive myofibroblast marker activated in a
cardiac tissue only during an injury. Postn activation leads to the expression of its
tamoxifen inducible cre which excises the stop codons in front of DTA and YFP reporter
genes. The expression of DTA toxin will inhibit protein synthesis in myofibroblasts and
make them undergo apoptosis (Ivanova et al., 2005). This is a great tool for in vivo cell
specific ablation as diphtheria toxin fragment A needs a receptor B to enter any other cell
type, making it toxic to only myofibroblasts where it is originally produced. The model for
control group had only Postn-Cre and YFP transgenes where the fibroblasts will express
YFP signal upon activation.

Figure 1.6b Mechanism of action of Isoproterenol drug on cardiomyocyte contractility
11

Isoproterenol used in this project is a well-characterized model of cardiac hypertrophy
as well as is easy and cost effective. It is a beta-adrenergic receptor agonist and an
established drug to induce heart failure. It promotes excess influx of calcium ions in the
sarcoplasmic reticulum which increases the contractility of cardiomyocytes and thus of
the whole heart muscle (Beta-Adrenoceptor Agonists – Richard E. Klabunde). This
calcium overload also causes cardiomyocyte death and in turn, fibrosis. The increased
heart rate and workload makes the heart tissue hypertrophic. Overstimulation of beta-
adrenergic receptor is one of the many ways that progresses to heart failure (Grimm et
al., 1998) (Krenek et al., 2009).

Hypothesis: Cardiac Myofibroblasts dysregulate angiogenesis in ISO-induced cardiac
hypertrophy.
Objective of Study:
1. To analyze the effect of in vivo myofibroblast ablation in an injured heart
2. To evaluate the role of fibroblasts and myofibroblasts in vessel formation – in vitro.

12

Chapter 2: Materials and Methods
2.1 DNA Genotyping:
Tail digestion is the most convenient DNA extraction method for genotyping. Tail
samples of 0.5 cm in length, were obtained by snipping the tip of the mouse tail in a
microfuge tube. 100µl of Direct Tail PCR Reagent and 1µl of Proteinase K was added per
sample and incubated at 55°C overnight. Next day, the digested samples in the microfuge
tubes were centrifuged at 5000 rpm for 5 mins and the supernatant to be used for PCR
reactions was transferred to a new tube. The samples can be stored in -20 for long term
use. Primers for PCR reaction of Periostin-Cre (Postn/Pn), YFP and DTA were ordered
from Integrated DNA Technologies and diluted at a concentration of 20µM before use.
PCR mix per sample:
Periostin

YFP DTA
GoTaq - 10µl GoTaq - 10µl GoTaq - 10µl
JOP - 1µl YFP - F - 0.7µl Forward Primer - 1µl
Forward Primer - 1µl YFP - WR - 0.7µl Reverse Primer - 1µl
Reverse Primer - 1µl YFP - MutR - 0.7µl -
dH2O - 6.5µl dH20 - 7.4µl dH2O - 7.5µl
DNA - 0.5µl DNA - 0.5µl DNA - 0.5µl

PCR Program:
Periostin YFP DTA
95.0°C - 3 mins
95.0°C - 30 secs
55.0°C - 45 secs 35 cycles
72.0°C - 45 secs
72.0°C - 5 mins
4.0°C - hold
94.0°C - 1 min
94.0°C - 1 min
59.0°C - 2 mins 35 cycles
72.0°C - 2 mins
72.0°C - 2 mins
4.0°C - hold

95.0°C - 2 mins
94.0°C - 1 min
68.0°C - 1 min 35 cycles
72.0°C - 1 min
72.0°C - 5 mins
4.0°C - hold

WT – 365 bp; Mut – 256 bp WT – 239 bp; Flox – 301bp Mut – 117 bp

13

2.2 Pump Implantation Surgery:
7-8 weeks old mice in control (Pn
MCM
;ROSA
YFP
) and experimental groups
(Pn
MCM
;ROSA
YFP
;ROSA
DTA
/+) were given tamoxifen food 1 week prior to the surgery and
it was continued until the day of tissue harvest. Surgical preparation and procedure are
elucidated below:

Materials Required:
Heating pad, Pad for surgery, Anesthesia setup, Isoflurane, Autoclaved surgical
instruments (scissors, forceps, clipper, suture clip remover), Autoclaved cotton swabs &
gauzes, Betadine (iodine), 70% Ethanol, Shaving machine, Buprenorphine, 1ml injection
syringe, 6-8 G injection needle (for buprenorphine), 27-1/2 + injection needle (for injecting
buprenorphine), Saline, Isoproterenol (covered with aluminum foil), Bead Sterilizer,
Surgical gloves, Marker pen, Eye ointment, Skin glue, Mini-pumps (Alzet osmotic pumps
with a reservoir volume of 100 µl) pump caps, Tape.

14

Surgical Procedure:
Body weight of mice were recorded with their ear tagged IDs on the day of surgery.
Isoproterenol amount to be administered was determined for each mouse based on their
body weight, as per the following calculation example: 60µg (per gram B.W. per day) X
25 (average B.W. of the mouse in gram) X 14 (number of days of drug delivery) X Twice
the number of mice the drug is required for = 60 X 25 X 14 X 4 (for 2 mice of 25g in
weight) = 0.084g Isoproterenol X 400ul of saline (100ul per pump). Surgical area was
prepared first following the aseptic guidelines. Mouse was anesthetized in a chamber at
3 units of isoflurane at an oxygen level of 1 unit. (One end of anesthesia chamber is
connected to the charcoal adsorber) 1.5 units of anesthesia was maintained throughout
the duration of shaving and surgery. The back of the mouse was shaved, and it was
injected with 0.1ml of buprenorphine subcutaneously. (The concentration of
buprenorphine as per the approved protocol should be 0.5 mg per kg body weight. Its
concentration in bottle is 0.5mg per ml.) Pumps were filled with 100µl of Isoproterenol or
saline as required and closed after making sure there were no bubbles inside the pump
cavity. Before operating, Optixcare ointment was applied on the eyes to prevent them
from drying and the shaved area was cleaned thrice with Betadine and 70% Alcohol in
turns using sterile cotton swabs. A small straight incision was made perpendicular to the
limb axis on the middle of the back and the pump was inserted subcutaneously in the
back pocket. The skin was closed with skin glue and sealed using suture clips for double
protection. The mouse was allowed to recover outside after removing it from anesthesia
and then transferred to an empty cage kept next to a heating pad. This procedure was
followed for all mice that were to be implanted with a pump. Surgery cards were filled
15

accurately, and the operated mice were observed twice daily for three days post-surgery.
Suture clips were removed 10 days after surgery.

2.3 Tissue Harvest and Processing:
The mice were sacrificed as per the IACUC guidelines, 14 days post-surgery and their
body weight was measured. The hearts were harvested, washed in ice cold PBS, weighed
and immersed in 4% PFA (dissolved in PBS) for 4 hours at 4°C. The whole hearts were
then cut longitudinally into two halves, such that both halves have left and right ventricles
and septum. These were then transferred into 5 ml Eppendorf tubes containing 30%
sucrose for overnight incubation at 4°C. Next day, they were embedded in separate
plastic molds into the OCT (Optimal Cutting Temperature) compound. The hearts were
placed in a chambers-facing-down orientation, so that the entire longitudinal plane of the
heart, falls on a single section. These cryo-blocks were frozen on dry ice and stored in -
80°C until further use.

2.4 Frozen Tissue Sectioning:
The frozen tissue blocks were cut using a cryostat to obtain 8µm thick sections on
poly-l-lysine coated glass slides. The slides were stored in -20°C until further use (Peters,
n.d.).

16

2.5 Histological Analysis:
2.5.1 CD31 Marker staining:
CD31 is an endothelial cell differentiation marker. It is expressed on the cell
membrane of endothelial cells present in both mature and newly formed blood vessels
(Gogiraju et al., 2019). Rat CD31 primary antibody (BD Biosciences) and Goat anti-Rat
594 secondary antibodies were used for immunostaining. Tissue sections were analyzed
under Leica Fluorescence Microscope. 5 images were captured per section per sample
from the left ventricular area, at 40X magnification.
2.5.2 YFP – αSMA co-staining:
In the mouse models being used, YFP is the reporter protein expressed upon
activation of Periostin-cre i.e. in myofibroblasts during an injury, whereas, αSMA (alpha-
smooth muscle actin), responsible for increased contractile activity, is a hallmark of
mature myofibroblasts. (Baum & Duffy, 2011) Mouse YFP and monoclonal (clone 1A4)
αSMA from Sigma were used as primary antibodies. Tissue sections were analyzed under
Leica Fluorescence Microscope. 5 images were captured per section per sample from
the left ventricular area, at 40X magnification.
2.5.3 Picrosirius Red staining:
Picrosirius Red stains collagen deposited within a tissue. This staining technique relies
on the birefringent properties of collagen. Left ventricular and septum regions were
captured using polarized phase contrast microscopy at 20X magnification. 10 images
were taken per section per sample to cover the entire area of interest. The images were
analyzed using ImageJ software.
17

2.6 Statistical Analysis:
Statistical significance was measured using student’s t test. All the calculations were
performed in excel using its formulae functions. The bar graphs were created in excel and
the error bars depicted standard deviation within each group.

2.1 Cell Culture:
HUVEC (human umbilical vein endothelial cells) P7 were thawed and cultured in M200
media with 2% LSGS and 1% Glutamax. The 10cm culture plates were coated with 1%
Gelatin (in dH2O) 15 mins prior to thawing / passaging cells. For passaging, the cells
were treated with 500X Trypsin. Fibroblasts (P16) were thawed and cultured in DMEM
media with 10% FBS and 1% PenStrep. To passage cells. 0.25% Trypsin was used. The
cells were suspended in 10% DMSO and 90% FBS to cryopreserve in liquid nitrogen for
future use.

2.8 Fibrin gel bead assay:

18

Figure 2.8a Schematic diagram of fibrin gel bead assay showing HUVEC and cytodex
bead suspension in a FACS tube as well as HUVEC coated beads embedded in fibrin gel
with fibroblasts seeded on top in a well of a 24-well plate.

Angiogenesis, a process of vessel formation from existing vessels, involves several
steps termed as endothelial cell sprouting, tube formation, branching and anastomosis.
Several in vitro assays have been developed to better understand this process and
investigate the factors regulating it. Fibrin gel bead assay is one such assay which bridges
the gap between simple monolayer cultures of endothelial cells and complex in vivo
assays available to study vessel development. It provides a 3-dimensional environment
similar to in vivo and facilitates real time imaging and gene expression studies in the most
efficient way (Nakatsu et al., 2007) (Cavallero et al., 2015) (Blüml, 2007).

Reagent Dilution:
Cytodex-3-Beads (10g/bottle): 0.5 g of dry beads were hydrated and swollen in 50ml
PBS (pH 7.4) for at least 3 hours on a shaker at room temperature. Beads were allowed
to settle down (~15 mins), supernatant was discarded, and beads were washed for a few
minutes in fresh PBS (50ml). The beads were again allowed to settle, old PBS was
discarded, and fresh PBS was added as per the desired quantity of beads per ml. i.e.
25ml PBS for → 20mg/ml → 60000 beads/ml; 50ml PBS for → 10mg/ml → 30000
beads/ml. The bead suspension was then transferred in a siliconized glass bottle
(windshield wiper or sigmacote) and autoclaved for 15 mins at 115°C. Finally, the bead
suspension was stored at 4°C until further use.
19

Aprotinin (10mg/bottle): Reconstituted lyophilized aprotinin at 4 U/ml in DI water.
Filtered and made aliquots of 1ml each. Stored at -20°C.
Fibrinogen Type I (1g in vial): Dissolved 2mg/ml fibrinogen in DPBS Note clottable
protein %. Heated in a 37°C water bath to dissolve the fibrinogen and mixed by inverting
the tube (not vortexing). Filtered through 0.22µm.
Thrombin (22mg = 1000units): Reconstituted in sterile water at 50 U/ml and made
aliquots of 0.5ml each. Stored at -20°C.

Coating Cytodex beads with HUVEC:
1ml of bead suspension was transferred into a 15ml tube and the beads were allowed
to settle. PBS was gently aspirated, and the beads were briefly washed with M200
medium (with 2% FBS + 1% Glutamax + 1% PenStrep). Beads were counted using a
hemocytometer by taking 2 aliquots of 10µl in volume (This was a fast step as beads
settle very quickly). As per the bead count calculation, required volume of suspension
was transferred in a separate FACS tube. HUVEC cells were then trypsinized and
centrifuged to obtain a trypsin free pellet. Cells were suspended in warm M200 media
and counted. The required volume based on the pre-established number of 400 HUVEC
cells per bead and 250 beads per well of a 24 well plate, was transferred to the FACS
tube containing beads. An additional 150µl of excess media was added to the tube and
slow pipetting was done 20 times to evenly mix the HUVEC-bead suspension. This tube
was then placed in a slanting position in the incubator at 37°C and gently shaken every
20 mins for 4 hours. After 4 hours, the bead suspension from the FACS tube was
transferred to a 6-well plate and was topped with 5ml of extra M200 media.
20

Figure 2.8b Image of a Cytodex-3 microcarrier bead coated with approx. 400 HUVEC
cells taken at 40X magnification under the light microscope.

Embedding Coated Beads in Fibrin Gel:
0.15 Units/ml of Aprotinin solution was added to the previously prepared fibrinogen
solution. The cell coated beads were transferred to a falcon tube and resuspended in
fresh M200 media after washing thrice by pipetting up and down slowly. The coated beads
were counted and resuspended in fibrinogen-aprotinin solution at a concentration of ~500
beads/ml (Fast step). 0.625 Units/ml of thrombin solution was first added to each well of
the 24-well pate (Adding thrombin first is important for clot formation). 0.5ml of fibrinogen-
aprotinin bead suspension was added in the middle of each well of a 24-well plate (the
pipette tip for each well was changed). The mixture was then pipetted to evenly distribute
the beads in the well. This plate was then incubated at 37°C for 10-15 mins to generate
a clot and then was seeded with ~20,000 fibroblasts per well. VEGF (20ng/ml) was added
21

per well to facilitate endothelial cell sprouting. Media was changed every alternate day
and images were captured daily under light microscope for 6 days.

Figure 2.8c Image of the fibrin gel embedded with HUVEC coated microcarrier beads,
taken under the light microscope 6 days post-gel embedding. Different stages of
anastomosis can be observed as such as endothelial cell sprouting, branching, tube
formation and anastomosis.

22

Chapter 3: Results

3.1 Reduced number of αSMA+YFP+ interstitial cells in DTA ISO group confirms
the deletion of myofibroblasts
The heart tissue sections of Pn
MCM
;ROSA
YFP
mice and Pn
MCM
;ROSA
YFP
;ROSA
DTA

mice were immuno-stained for YFP and myofibroblast marker αSMA. Number of
myofibroblasts were counted based on co-expression of YFP and αSMA signals. Control
saline-infusion (Cont Saline) hearts had negligible number of myofibroblasts present due
to the absence of injury. In the ISO-infusion group (Cont ISO), isoproterenol induced injury
resulted in significantly higher number of myofibroblasts as indicated by positive staining
of YFP and αSMA. The heart samples from ISO-infused DTA group had significantly lower
number of myofibroblasts as compared to the control injury group (Cont ISO) without
DTA. This data confirmed the efficiency of DTA toxin in ablating myofibroblasts.

Figure 3.1 Left panel shows representative images of YFP and αSMA co-stained tissue
sections from all four groups. The right panel shows the bar graph of average co-localized
signals in each group. Statistical significance is shown with *: p<0.05 and #: p<0.01
23

3.2 Myofibroblast ablated hearts have reduced perivascular and interstitial fibrosis
The saline and ISO treated hearts from control and experimental groups were
sectioned and stained with Picrosirius Red to visualize collagen deposition within the
heart. The ISO injury model (Cont ISO) induced large amount of collagen deposition in
the perivascular and interstitial region of the cardiac tissue. The DTA ISO group showed
significantly less collagen deposition despite the injury, since collagen secreting
myofibroblasts were efficiently ablated. Both YFP and DTA mice treated with saline
showed negligible collagen deposition as expected.

Figure 3.2 The figure shows representative images of collagen deposition within
perivascular and interstitial areas of each group, stained with picrosirius red stain.

24

3.3 Vessel number increases in myofibroblast ablated hearts
The heart tissue sections of Pn
MCM
;ROSA
YFP
mice and Pn
MCM
;ROSA
YFP
;ROSA
DTA

mice were immuno-stained for CD31, an endothelial marker. I observed that the DTA ISO
group with ablated myofibroblasts, showed significantly higher vessel density as
compared to the Cont ISO group with activated fibroblasts. There was a significant
difference between the control groups as expected, with YFP Saline showing higher
vessel density as compared to YFP ISO. Each dot represents one vessel.

Figure 3.3 Left panel shows representative images of CD31 stained tissue sections from
all four groups. The right panel shows the bar graph depicting vessel numbers per field
of each group. Statistical significance is shown with #: p<0.01

25

3.4 Lower Heart weight / Body weight ratio in Myofibroblast ablated hearts depict
lesser hypertrophy
Heart weight / Body weight ratio is a standard method for measuring cardiac
hypertrophy, where higher ratio associates with hypertrophy. The data was collected from
a new set of mice incorporating ROSA
Td
as the reporter instead of YFP. The heart weight
/ body weight ratios (HW/BW) of isoproterenol treated Pn
MCM
;ROSA
Td
mice were
significantly higher than that of as compared to that of saline treated Pn
MCM
;ROSA
Td
mice.
But the DTA ISO group with ablated myofibroblasts showed significantly lower HW/BW
ratio than the Cont ISO group. We also observed that DTA saline group had significantly
higher HW/BW ratio than the saline-treated control group, which may arise from toxic
effects of DTA transgene.

Figure 3.4 Bar graph representing heart weight / body weight ratios of each group.
Statistical significance is shown as *: p<0.05 and #: p<0.01
26

Discussion

The term angiogenesis refers to the formation of new vessels from existing
vasculature. When cardiac tissue undergoes any kind of injury that leads to loss of
cardiomyocytes, the surviving myocytes increase in size to make up for the loss of
function. This increase in cell area and enlargement of cardiac muscle tissue is known as
hypertrophy. Pathological hypertrophy is a compensatory mechanism of a failing heart as
opposed to the physiological hypertrophy that occurs in a developing heart or during
exercise. The walls of the heart muscle keep getting thicker to sustain the increase in
workload of supplying enough blood to the body, despite the injury. This increased growth
of heart muscle is not always backed up by the proportional increase in angiogenesis,
leading to decreased oxygen and nutrient supply to each cardiomyocyte. Angiogenic
insufficiency along with increased distance between capillaries surrounding each
hypertrophied myocyte, advances the heart failure (de Graaf et al., 2014).
Literature review suggests enhancement of angiogenesis in a failing heart would
improve cardiac function and prolong the end stage of heart failure. Recent studies have
described the role of several factors expressed by cardiomyocytes, endothelial cells as
well as inflammatory cells and pericytes in endothelial cell sprouting and vessel formation.
Clinically, upregulating these factors could ameliorate the condition of angiogenesis but
also showed pro-hypertrophic or pro-fibrotic response within the cardiac tissue (Gogiraju
et al., 2019). Thus, further investigations are needed to find a promising target for
augmenting angiogenesis.
27

Here, we examined the role of resident and activated cardiac fibroblasts in regulating
angiogenesis. We hypothesized that activated fibroblasts, also called as myofibroblasts,
dysregulate angiogenesis and ablating these cells would improve the angiogenic ability
of endothelial cells to sprout and form vessels. Mouse models were generated with
transgenic DTA to specifically delete myofibroblasts in vivo. Isoproterenol drug was used
to make the heart muscle work harder and stimulate the activation of fibroblasts. The
hearts were harvested 14 days post the isoproterenol filled osmotic pump implantation
and the tissues were processed for further analysis.
It was found that the vessel number in injured hearts with ablated myofibroblasts was
significantly higher than in the injured hearts where myofibroblasts were not deleted. To
confirm that our DTA model was efficient in deleting myofibroblasts, I immuno-stained the
tissue sections for a myofibroblast marker and found that they were adequately deleted
by the DTA toxin as compared to the injury model without DTA expression. To further
evaluate the robustness of the model and the effect of Isoproterenol drug, I analyzed
collagen deposition within the tissue using picrosirius red stain and observed that the
injury model had high amounts of collagen deposited by the activated fibroblasts within
interstitial and perivascular areas than in the myofibroblast ablated hearts. Thus, it can
be concluded from these experiments that, myofibroblasts have some role in
dysregulating angiogenesis. To validate the results, more samples should be added in
each group and several consecutive sections must be analyzed for each parameter of
interest.
28

Future direction of this project would be to further investigate the mechanistic role of
fibroblasts and myofibroblasts in angiogenesis using fibrin gel bead assay. This assay
was set up in the lab and the experimental design is explained in the figure below.

The stepwise process of angiogenesis in vitro can be imaged over a period of 6-10
days. Difference between fibroblast and myofibroblast influence on endothelial cell
sprouting, lumen formation, branching and anastomosis can be analyzed using light
microscopy and ImageJ software (Cavallero et al., 2015). The factors expressed by
these two cell types can be evaluated with Mass spectrometry. Apart from this, the
viability of the endothelium should be tested and endothelial cell integration into
functional vasculature will be tested using IB4 staining.
Another interesting result observed from the experiments was that the injured hearts
with ablated myofibroblasts showed lesser hypertrophy than the injury group without
myofibroblast ablation. This result was obtained using the heart weight / body weight
ratio which can be confirmed by measuring area of individual cardiomyocytes with WGA
staining technique.
29

References

Alex, L., & Frangogiannis, N. G. (2018). The cellular origin of activated fibroblasts in the
infarcted and remodeling myocardium. Circulation Research, 122(4), 540–542.
https://doi.org/10.1161/CIRCRESAHA.118.312654
Baum, J., & Duffy, H. S. (2011). CityPaws Animal Hospital 1823. Journal of
Cardiovascular Pharmacology, 57(4), 376–379.
https://doi.org/10.1097/FJC.0b013e3182116e39.Fibroblasts
Blüml, G. (2007). Microcarrier Cell Culture Technology. 149–178.
https://doi.org/10.1007/978-1-59745-399-8_5
Cavallero, S., Shen, H., Yi, C., Lien, C. L., Kumar, S. R., & Sucov, H. M. (2015).
CXCL12 Signaling is Essential for Maturation of the Ventricular Coronary
Endothelial Plexus and Establishment of Functional Coronary Circulation.
Developmental Cell, 33(4), 469–477. https://doi.org/10.1016/j.devcel.2015.03.018
Davis, J., & Molkentin, J. D. (2014). Myofibroblasts: Trust your heart and let fate decide.
Journal of Molecular and Cellular Cardiology, 70, 9–18.
https://doi.org/10.1016/j.yjmcc.2013.10.019
de Graaf, W. L., Vandoorne, K., Arslan, F., Nicolay, K., & Strijkers, G. J. (2014).
Contrast-Enhanced T1-Mapping MRI for the Assessment of Myocardial Fibrosis.
Current Cardiovascular Imaging Reports, 7(4), 1–12.
https://doi.org/10.1007/s12410-014-9260-6
Duncker, D. J., Koller, A., Merkus, D., & Canty, J. M. (2015). Regulation of coronary
blood flow in health and ischemic heart disease. Progress in Cardiovascular
Diseases, 57(5), 409–422. https://doi.org/10.1016/j.pcad.2014.12.002
Gogiraju, R., Bochenek, M. L., & Schäfer, K. (2019). Angiogenic Endothelial Cell
Signaling in Cardiac Hypertrophy and Heart Failure. Frontiers in Cardiovascular
Medicine, 6(March). https://doi.org/10.3389/fcvm.2019.00020
González, A., Schelbert, E. B., Díez, J., & Butler, J. (2018). Myocardial Interstitial
Fibrosis in Heart Failure: Biological and Translational Perspectives. Journal of the
American College of Cardiology, 71(15), 1696–1706.
https://doi.org/10.1016/j.jacc.2018.02.021
Grimm, D., Elsner, D., Schunkert, H., Pfeifer, M., Griese, D., Bruckschlegel, G., Muders,
F., Riegger, G. A. J., & Kromer, E. P. (1998). Development of heart failure following
isoproterenol administration in the rat: Role of the renin-angiotensin system.
Cardiovascular Research, 37(1), 91–100. https://doi.org/10.1016/S0008-
6363(97)00212-5
Grossman, W. (1980). Cardiac hypertrophy: Useful adaptation or pathologic process?
The American Journal of Medicine, 69(4), 576–584. https://doi.org/10.1016/0002-
9343(80)90471-4
30

Herum, K., Lunde, I., McCulloch, A., & Christensen, G. (2017). The Soft- and Hard-
Heartedness of Cardiac Fibroblasts: Mechanotransduction Signaling Pathways in
Fibrosis of the Heart. Journal of Clinical Medicine, 6(5), 53.
https://doi.org/10.3390/jcm6050053
Ivanova, A., Signore, M., Caro, N., Greene, N. D. E., Copp, A. J., & Martinez-Barbera, J.
P. (2005). In vivo genetic ablation by Cre-mediated expression of diphtheria toxin
fragment A. Genesis, 43(3), 129–135. https://doi.org/10.1002/gene.20162
Kaur, H., Takefuji, M., Ngai, C. Y., Carvalho, J., Bayer, J., Wietelmann, A., Poetsch, A.,
Hoelper, S., Conway, S. J., Möllmann, H., Looso, M., Troidl, C., Offermanns, S., &
Wettschureck, N. (2016). Targeted Ablation of Periostin-Expressing Activated
Fibroblasts Prevents Adverse Cardiac Remodeling in Mice. Circulation Research,
118(12), 1906–1917. https://doi.org/10.1161/CIRCRESAHA.116.308643
Krenek, P., Kmecova, J., Kucerova, D., Bajuszova, Z., Musil, P., Gazova, A.,
Ochodnicky, P., Klimas, J., & Kyselovic, J. (2009). Isoproterenol-induced heart
failure in the rat is associated with nitric oxide-dependent functional alterations of
cardiac function. European Journal of Heart Failure, 11(2), 140–146.
https://doi.org/10.1093/eurjhf/hfn026
Mensah, G. A., & Brown, D. W. (2007). An overview of cardiovascular disease burden in
the United States. Health Affairs, 26(1), 38–48.
https://doi.org/10.1377/hlthaff.26.1.38
Nakatsu, M. N., Davis, J., & Hughes, C. C. W. (2007). Optimized fibrin gel bead assay
for the study of angiogenesis. Journal of Visualized Experiments, 3, 2–3.
https://doi.org/10.3791/186
Opie, L. H. (1990). Compensation and overcompensation in congestive heart failure.
American Heart Journal, 120(6 PART 2), 1552–1557. https://doi.org/10.1016/0002-
8703(90)90058-6
Peters, S. R. (n.d.). A Practical Guide to Frozen Section Technique.
Roger, V. L. (2013). Epidemiology of heart failure. Circulation Research, 113(6), 646–
659. https://doi.org/10.1161/CIRCRESAHA.113.300268
Travers, J. G., Kamal, F. A., Robbins, J., Yutzey, K. E., & Blaxall, B. C. (2016). Cardiac
fibrosis: The fibroblast awakens. Circulation Research, 118(6), 1021–1040.
https://doi.org/10.1161/CIRCRESAHA.115.306565

Abstract (if available)

Abstract Diseases like hypertension, myocardial ischemia, diabetes, congenital heart defect etc. worsen with time and make the heart work harder for it to supply enough blood throughout the body. This excess workload causes injury to the cardiac tissue and the heart muscle undergoes hypertrophy to compensate for its loss of function. The pathological cardiomyocyte growth demands increase in nutrient and oxygen supply which is not always backed up by the proportional increase in angiogenesis. This lack of vascularization worsens cardiac remodeling and tissue contractility during injury and accelerates the progression to heart failure. Recent studies have found the paracrine roles of cardiomyocytes, pericytes and inflammatory cells in (dys)regulating angiogenesis during cardiac hypertrophy, but little is known about the role of cardiac fibroblasts in this process. Cardiac fibroblasts are responsible for providing support and maintaining the structure of the heart tissue by secreting extracellular matrix (ECM) proteins. These resident fibroblasts become activated and transform into myofibroblasts to secrete excess ECM proteins during the wound repair process post injury (Travers et al., 2016). ❧ My project focuses on understanding the role of resident and activated fibroblasts in modulating angiogenesis during cardiac injury. I will be using in vivo and in vitro models to investigate this question.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

TNNI3K expression affects nucleation of cardiomyocytes and skeletal myoblasts

PDF

Characterization of Cxcr7 in zebrafish cardiac lymphatic vessel development

PDF

The roles of Tnni3k in heart regeneration, cardiac conduction system defects and cardiomyopathy

PDF

A novel role for post-migratory neural crest cells in cardiac outflow tract alignment: insights into the molecular basis of concomitant congenital cardiac and cleft malformations

PDF

PRMT1 controls subcellular localization of p53 via PARP1

PDF

Characterization of metabolically distinct muscle resident fibro-adipogenic subpopulations reveals a potentially exploitable mechanism of skeletal muscle aging and disease

PDF

Zebrafish as a blueprint for cardiac regeneration

PDF

The effect of urinary bladder matrix on irradiated wound healing

PDF

Tools to study the epicardium's response during cardiac regeneration

PDF

The role of Runx2 in the development of the tendon-bone attachment unit

PDF

Engineering a post-infarct human myocardium on a chip to reveal oxygen-dependent crosstalk in cardiac fibroblasts and myocytes

PDF

Proliferation and maturation events in second heart field cells during cardiovascular development activated by the Delta like ligand-4 mediated notch signaling

PDF

Renal extracellular matrix bioactivity in a range of physiological states: novel insights for kidney regeneration

PDF

The differential effects of selective COX-2 inhibitors on cell proliferation and induced ER stress in glioblastoma and pancreatic carcinoma cell lines

PDF

Bimodal effects of bone morphogenetic proteins in prostate cancer

PDF

The role of fibroblast growth factor signaling on postnatal hepatic progenitor cell expansion

PDF

Characterization of a new chromobox protein 8 (CBX8) antagonist in a model of human colon cancer

PDF

Quantitative evaluation of the effectiveness of an integrin antagonist, vicrostatin, in cell migration

PDF

The response of Allograft inflammatory factor-1 to neurotoxic injury, and its role as a secreted protein

PDF

The role of endoplasmic reticulum proteins GRP78 and IP3R1 in regulation of glucose homeostasis and acute pancreatitis

Asset Metadata

Creator Upadhyay, Aesha (author)

Core Title Investigating the role of cardiac fibroblasts in modulating angiogenesis during heart injury

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 08/04/2020

Defense Date 06/03/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag angiogenesis,DTA,fibroblast,fibrosis,heart failure,hypertrophy,myofibroblast,OAI-PMH Harvest,periostin,ventricle

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Xu, Jian (committee chair), Hong, Young-Kwon (committee member), Lien, Ching-Ling (committee member)

Creator Email aeshadiu@usc.edu,upadhyayaesha@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-358712

Unique identifier UC11666551

Identifier etd-UpadhyayAe-8880.pdf (filename),usctheses-c89-358712 (legacy record id)

Legacy Identifier etd-UpadhyayAe-8880.pdf

Dmrecord 358712

Document Type Thesis

Rights Upadhyay, Aesha

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA